To assess the impact of a single esketamine intravenous (IV) injection on the quality of recovery during early period after modified radical mastectomy for breast cancer.0), one (T1) and two hours after the extubation (T2), the 40-item Quality of Recovery (QoR-40) scores one (D1) and two days after the surgery (D2), and the rate of adverse reactions were assessed in both groups.This retrospective study included 80 patients who underwent modified radical mastectomy under general anesthesia in Huzhou Maternity & Child Health Care Hospital from March to October 2022. All patients were between 35 to 55 years, weighting between 45 and 70 kg and Grade-I or II according to the American Society of Anesthesiologists (ASA). Patients were grouped based on the type of pain management used. Patients (n=40) who were given 0.25 mg/kg esketamine single IV injection prior to completion of the surgery were assigned to Group-E, and patients (n=40) who were not treated with esketamine, comprised the control Group-C. Patients’ data, such as education years, operation time, blood loss, the 9-item Quality of Recovery (QoR-9) scores before the anesthesia induction . Similarly, the QoR-40 scores on D1 and D2 in Group-E were significantly higher compared to Group-C (P<O.05).Compared with TSingle intravenous injection of esketamine can improve the quality of the recovery of breast cancer patients during the early period after the modified radical mastectomy. Modified radical mastectomy via axillary lymph node dissection preserves patient’s pectoralis major and minor muscles and medial and lateral pectoral nerves. This type of surgery is currently the most commonly used surgical procedure for breast cancer.5Ketamine has a positive effect in pain management and mood improvement.This retrospective study was performed in Huzhou Maternity & Child Health Care Hospital. The target population was patients who underwent elective modified radical mastectomy for breast cancer in our hospital and consented to participate in the study. Patients were retrospectively allocated into two groups, Group-E and Group-C, n=40 each, based on the intraoperative pain management. A total of 80 ASA grade I-II patients, aged 35-55 years, weighting 45-70 kg, were included. Patients in Group-E were given 0.25 mg/kg esketamine single IV injection prior to completion of the surgery, while patients in Group-C did not receive the drug.ASA grade I-II35-55years, 45-70 kgUnderwent elective modified radical mastectomy for breast cancer Complete clinical dataHistory of psychosis.Hypertension.Hyperthyroidism.History of allergy or addiction to narcotic drugs.Severe insufficiency of vital organs such as liver and kidney. Incomplete medical recordsThis study got the approval of the ethics committee of the Huzhou Maternity & Child Health Care Hospital . Signed informed notification form was obtained from the participating patients.2), electrocardiogram (ECG) and electroencephalographic bispectral index (BIS) was done. General anesthesia was induced by the sequential administering of 0.03 mg/kg midazolam, 0.2 mg/kg etomidate, 0.3 μg sufentanil/kg, 0.15 mg/kg cis-atracurium. Mechanical ventilation was maintained after the intubation. Intraoperatively, Propofol 4~6 mg·kg-1·h-1 and Remifentanil 0.1~0.3 μg·kg-1·h-1 were infused intravenously, and additional skeletal muscular relaxants were intermittently administered to maintain anesthesia with PETCO2 35~45mmHg, BIS 40~60.Patients were admitted to the operating room and peripheral veins access was established. Continuous monitoring of blood pressure (BP), pulse oximetry  was 2.0 ml/time with an interval of 15 minutes.0), one hour after the extubation (T1) and two hours after the extubation (T2). The QoR-9 score is used to measure the quality of recovery after general anesthesia, and contains nine items, ranging from the score zero to two, with a total score of 18. Higher scores indicate better quality of patients’ recovery from the general anesthesia.1) and day two (D2) for both groups to assess quality of patients’ recovery after the surgery. The QoR-40 score includes 40 questions in five areas, such as physical comfort, emotional state, self-care, psychological support, and pain. Each question has a score of one to five. The total score ranges from 40 to 200. Greater total score indicates better recovery quality.Patient’s data, such as years of education, duration of surgery, and intraoperative blood loss was recorded for both groups. Record of QoR-9 scores of all patients was done before the initiation of anesthesia (Tx±s). The statistical data were tested by χ2 test or Fisher’s exact probability method. P < 0.05 was statistically significant.Analysis was done by SPSS 20.0 statistical software. Intergroup comparison of the normal distribution data was done by t-test and presented as mean ± standard deviation ( 1 and D2 compared to Group-C (P<0.05) (As summarized in (P<0.05) . The rat(P<0.05) .The primary finding of this study is that single intravenous injection of esketamine can improve the quality of recovery of breast cancer patients during the early postoperative period after the modified radical mastectomy.In the current study, the QoR-9 and QoR-40 scores are designed as the primary outcome measure. QoR-9 scores system is a nine-item patient-rated score of self-perceived comfort that assesses the patient’s quality of recovery after the general anesthesia using the following criteria: anxiety and fear; level of need for support from others; level of understanding of instructions; level of simple living; level of gastrointestinal discomfort and self-toileting; level of ease of breathing; level of headache, back pain and muscle pain; level of nausea and vomiting; and level of severe pain or persistent moderate pain.Modified radical mastectomy for breast cancer can have both physical and psychological impact on the patient, as it is associated with considerable postoperative pain and emotional distress due to cosmetic defects. Studies show that patient’s distress may significantly affect wound healing.1 and T2 as opposed to the control group. The QoR-9 scores of patients in Group-E were significantly elevated, suggesting that a single subanesthetic dose of esketamine could significantly improve patients’ comfort levels during the early postoperative stage, probably due it its anti-inflammatory and analgesic effect. A single subanesthetic dose of esketamine was associated with increased patient self-perceived comfort after the surgery.Patients that were administered esketamine (Group-E) had better self-perceived comfort, need for support from others, simple living, and pain-related scores at TOur results are consistent with the observations of Cao et al.211 and D2 were markedly greater in Group-E patients compared to the patients in the control group.We have observed that the pain scores and physical comfort scores in the QoR-40 at D1 and D2 were significantly greater in Group-E compared to Group-C patients, as indicated by better QoR-40 scores. The final QoR-40 total score was also markedly higher in Group-E patients.Previous studies also demonstrated the marked antidepressant effect of subanesthetic doses of ketamine.The common adverse effects of esketamine include increased BP and HR, psychiatric symptoms such as anxiety, nightmares, hallucinations, and dissociation, as well as nausea and vomiting. However, these adverse effects are transient and dose-dependent.This is a retrospective study with a small sample size. Further prospective studies with larger sample sizes are needed to confirm our results. Only 0.25 mg/kg of esketamine was administered to the patients. Further studies are needed to determine whether this is the optimum dose. Second, esketamine was given before the extubation, 10 min before the end of the surgery. Further studies should determine the optimal timing for the administration of esketamine.A single postoperative intravenous injection of esketamine is simple and easy to administer and can lead to significantly improved quality of early recovery after breast cancer surgery. Single postoperative IV injection of esketamine, thus, may be safely used in clinical practice.GF: Conceived and designed the study.WY and HY: Collected the data and performed the analysis.GF: Was involved in the writing of the manuscript and is responsible for the integrity of the study.All authors have read and approved the final manuscript.


There have been few reports on the diagnostic performance of soluble interleukin-2 receptor (sIL-2R) for lymphoma. A cross-sectional study was conducted at a university hospital; all patients who were admitted to the Division of General Internal Medicine and underwent serum sIL-2R testing were included. Patients were divided into two groups based on the presence of fever (≥ 38.0 °C). Among 602 patients, 421 had fever and 76 were diagnosed with lymphoma (48 of the 76 were in the febrile group). In all patients, the area under the receiver operating characteristic curve (AUROC) of sIL-2R for the diagnosis of lymphoma was 0.81 . The AUROC was significantly higher in the febrile group  than in the afebrile group . In the febrile group, the sensitivity and specificity were 81.2% and 82.3%, respectively, with an optimal cutoff value of 3,250 U/mL. In the afebrile group, they were 89.3% and 54.9%, respectively, with a cutoff value of 868 U/mL. Serum sIL-2R showed high performance as an adjunctive diagnostic marker for lymphoma, particularly among febrile patients. Different cutoff values should be used for patients with and without fever to maximize diagnostic performance. It mainly promotes proliferation of both CD4+ and CD8+ T cells. An IL-2 receptor is composed of three subunits. Soluble interleukin-2 receptor (sIL-2R), which is the cleaved chain of the IL-2 receptor alpha  on the cell membrane due to proteolytic processing, has been recognized as a biomarker of T-cell activation. sIL-2R is considered a tumor-related biomarker of lymphoma. Several studies have reported an association between pretreatment sIL-2R levels and progression free survival in diffuse large B-cell lymphoma and follicular lymphoma3. sIL-2R has also been evaluated as a prognostic marker after treatment among patients with diffuse large B-cell lymphoma and follicular lymphoma5.Interleukin 2 (IL-2) is a significant cytokine that regulates T-cell responses6. Despite this fact, in Japan, sIL-2R is widely measured in patients suspected to have lymphoma. Moreover, to the best of our knowledge, there have been no reports regarding the impact of sIL-2R in patients with inflammation. The performance of sIL-2R as a diagnostic tool can change in patients with inflammation because of sIL-2R levels due to T-cell activation caused by inflammatory diseases other than lymphoma. This study evaluated the diagnostic performance of sIL-2R for lymphoma in patients with and without fever.However, there have been few studies on the performance of sIL-2R as a diagnostic tool for lymphomaThis cross-sectional study used medical records from the Jichi Medical University Hospital . All patients (> 15 years old) admitted to the Division of General Internal Medicine between January 2014 and June 2021 and tested for serum sIL-2R anytime from 7 days prior to admission till discharge were included in this study. Hospitalized patients who already had a confirmed diagnosis of lymphoma and were tested for sIL-2R for follow-up purposes (non-diagnostic purposes) were excluded from the study. If a patient had multiple eligible episodes during the study period, only the first episode was included.We divided the enrolled patients into two groups based on the presence of fever: febrile and afebrile. Patients who had an axillary temperature of 38.0 °C or higher from the onset of illness to the third day of hospitalization were defined as febrile patients. If patients reported having fever prior to admission without information on whether they met the definition of fever, they were classified into the febrile group if the word "high-grade fever" was mentioned in their medical records.Information extracted from medical records included patient age, sex, history of chronic hemodialysis, and final diagnosis that caused eligible hospitalization. We included only those patients who were histologically confirmed to have lymphoma in the final diagnosis of lymphoma; we reviewed lymphoma subtypes based on histopathological reports. The results of laboratory tests were also collected: white blood cell (WBC) and platelet (PLT) counts as well as levels of hemoglobin, serum sIL-2R, C-reactive protein (CRP), lactate dehydrogenase (LDH), ferritin, and erythrocyte sedimentation rate. The serum concentration of sIL-2R was measured using a chemiluminescent enzyme immunoassay , with a reference range of 154–474 U/mL.This study was conducted in accordance with the Declaration of Helsinki. The Ethics Committee of Jichi Medical University Hospital approved this study and waived written informed consent because of the retrospective design .The primary outcome of interest was the performance of sIL-2R in the diagnosis of lymphoma in patients with and without fever. A receiver operating characteristic (ROC) curve was used to determine the diagnostic accuracy and cut-off values of serum sIL-2R levels. The ROC curve of the febrile group was compared with that of the afebrile group. The sensitivity and specificity of the sIL-2R levels for diagnosis were also calculated.P-values were two-tailed; P-values < 0.05 were considered statistically significant. All statistical analyses were performed using R .Continuous variables are presented as medians and interquartile ranges (IQRs) and compared using the Wilcoxon signed-rank test. Categorical variables were compared using McNemar’s chi-square test. The sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio of sIL-2R, WBC, PLT, and LDH levels for the diagnosis of lymphoma were determined. The area under the ROC curve (AUROC) was measured to estimate diagnostic performance, and the optimal cutoff values were determined using Youden’s index. The ROC curves of the febrile group were compared with those of the afebrile group using the DeLong test. All 8. Patient characteristics are shown in Table A total of 625 patients were included in this study. We excluded 23 patients on chronic hemodialysis from further analysis because serum sIL-2R levels during hemodialysis can be elevated regardless of the presence of lymphoma or other diseaseP < 0.001). In the febrile group, the median sIL-2R levels were significantly higher in patients with lymphoma than in those without lymphoma . In the afebrile group, similar significant trends with relatively low sIL-2R levels were observed; the median sIL-2R levels were 1,895 U/mL  in afebrile patients with lymphoma and 797 U/mL  in those without lymphoma. In the febrile group, WBC and PLT counts were significantly lower, and monocyte percentage, monocyte-to-lymphocyte ratio, and LDH levels were significantly higher in patients with lymphoma than in those without lymphoma, although there was no significant difference between patients with and without lymphoma in the afebrile group  were diagnosed with lymphoma (48 in the febrile group and 28 in the afebrile group). The median serum sIL-2R level was significantly higher in patients with lymphoma than in those without lymphoma  was 0.88 (0.81–0.94) in the febrile group (P < 0.001) and 0.75 (0.65–0.85) in the afebrile group (P < 0.001). The AUROC was significantly higher in the febrile group than in the afebrile group (P = 0.044). The sensitivity and specificity were 81.2% and 82.3%, respectively, with an optimal sIL-2R cutoff value of 3,250 U/mL in the febrile patients, and 89.3% and 54.9%, respectively, with an optimal cutoff value of 868 U/mL in afebrile patients. The diagnostic performance  with different cut-off values is shown in Table The ROC curves for the prediction of lymphoma by analyzing sIL-2R levels as well as other variables  are shown in Fig. AUROC values (95% CI) of the other variables in all patient cohorts were as follows: age, 0.57 0.51–0.64); WBC count, 0.60 (0.53–0.67); monocyte-to-lymphocyte ratio, 0.62 (0.55–0.69); PLT count, 0.64 (0.58–0.71); and LDH level, 0.69 (0.63–0.75). The AUROC values of the PLT count and LDH level in the febrile group were significantly greater than those in the afebrile group  of patients with lymphoma who initially presented with fever of unknown origin had aggressive lymphoma and showed poorer performance status, poorer prognosis, and lower complete remission rate than patients with lymphoma without a history of fever of unknown origin6. Their overall results were similar to our results for the afebrile group, mainly because their study included a small fraction of febrile patients . Studies examining the utility of sIL-2R in the diagnosis of lymphoma are limited; Higashi et al. analyzed the values of sIL-2R in patients who underwent random skin biopsy for the diagnosis of intravascular lymphoma and suggested that high levels of sIL-2R may be one of the diagnostic indicators of the disease10.The AUROC of sIL-2R in afebrile patients (0.75) was lower than that in febrile patients (0.88). Murakami et al. evaluated the diagnostic performance of 248 adult patients who were suspected to have lymphoma and had serum sIL-2R tested at a Japanese university hospital. They reported that the AUROC, sensitivity, and specificity were 0.695, 35%, and 93%, respectively, when the sIL-2R cutoff was set at 1,950 U/mL13. Elevation of sIL-2R levels in patients with hypereosinophilic syndrome has also been reported in previous studies15. These observations are consistent with our study in which 6 of the 15 patients with non-lymphoma-associated hemophagocytic lymphohistiocytosis and three of the seven patients with hypereosinophilic syndrome had sIL-2R levels above 3250 U/mL.Although serum sIL-2R levels showed good discriminating power in febrile patients in our study, the role of sIL-2R testing in the diagnosis of lymphoma is complementary, and false positives and negatives should be considered. The levels of sIL-2R are elevated in hematologic diseases other than lymphoma. For example, hemophagocytic lymphohistiocytosis is associated with sIL-2R elevation and is considered a marker for disease activity and prognosis19. In the present study, the sIL-2R levels in 43% (6/14) of patients with tuberculosis and 75% (3/4) of patients with rickettsial infections were > 3250 U/mL; therefore, we should consider these diseases in addition to lymphomas when sIL-2R levels are elevated in patients with fever of unknown origin. Eighteen patients in this study were diagnosed with having experienced drug reactions; among them, 12 exhibited sIL-2R levels of 2000 U/mL or higher. Several studies have reported elevated sIL-2R levels among patients with drug-induced adverse events21. Kluge et al. measured cytokine levels in patients who started clozapine and reported that the levels of interleukin-6 and sIL-2R were increased in patients who developed drug-induced fever, suggesting that drug reactions may activate inflammatory cytokines, resulting in the elevation of sIL-2R levels20.In addition to hematologic disorders, sIL-2R elevation has been observed in various autoimmune, inflammatory, and infectious diseases, including systemic lupus erythematosus, adult-onset Still’s disease, sarcoidosis, tuberculosis, and rickettsial infectionsIn a real-world setting, some patients do not obtain a final diagnosis for a variety of reasons, such as the patients or their households do not wish to undergo invasive examination, or the patient's critically-ill condition does not allow for evaluation. In this study, there were four patients who were strongly suspected to have lymphoma but this was not confirmed histologically; we categorized these patients into the non-lymphoma group . However, the diagnostic performance remained approximately the same when they were categorized into the lymphoma group: the AUROC (95% CI) was 0.81 (0.75–0.87) in all patients, 0.88 (0.81–0.94) in the febrile group, and 0.75 (0.65–0.85) in the afebrile group.8. A preliminary, unpublished subgroup analysis from our study showed that the median serum sIL-2R level of patients without lymphoma on chronic hemodialysis (n = 23) was significantly higher than that of non-lymphoma patients not on hemodialysis: 3880 U/mL  vs. 1230 U/mL  in the febrile group and 1720 U/mL  vs. 797 U/mL  in the afebrile group. In our study, none of the patients with lymphoma underwent chronic hemodialysis. Further independent studies are warranted for patients on hemodialysis to assess the diagnostic performance of serum sIL-2R in lymphoma.We excluded patients on chronic hemodialysis from our study because sIL-2R levels may be elevated by factors other than active disease. The mechanism is thought to involve a combination of factors, including decreased renal clearance and increased production of sIL-2R due to exposure to cytokine elicitors in the dialysate solutionThe present study has several limitations. First, this was a single-center study that enrolled patients who were hospitalized at the Division of General Internal Medicine. Therefore, there may have been a bias in the patient population. The participants of this study were predominantly older patients, and it is uncertain whether our results can be generalized to younger patients. Although patients who did not require hospitalization for diagnosis and those who were referred to our hospital with a confirmed diagnosis of lymphoma were not included, the results of our study may provide important insight into estimating the likelihood of lymphoma for a population that is hospitalized for examination of fever. Second, we defined fever as an axillary temperature of ≥ 38.0 °C. The definition of fever can vary across studies, and the degree of fever can be affected by medications, such as antipyretics. Third, this study included patients for whom a final diagnosis could not be obtained even after close examination or for whom further examination was not attempted based on their condition or willingness. Therefore, some cases of lymphoma may be misclassified as non-lymphoma. Fourth, patients diagnosed with lymphoma might have a certain simultaneous complication  that can affect sIL-2R levels, which may have affected the performance of sIL-2R for the diagnosis of lymphoma. Finally, this retrospective study used medical records, which limited the reliability of the information.In conclusion, this study showed that serum sIL-2R levels are a useful adjunctive diagnostic tool for lymphoma, particularly in febrile patients. To maximize the sensitivity and specificity for lymphoma, cutoff values of serum sIL-2R should be categorized based on the presence or absence of fever. The optimal cutoff levels for patients on hemodialysis remains a subject for future research.Supplementary Figure 1.Supplementary Legends.


Here, the authors highlight a recently proposed luminescent enhancement mechanism and record-breaking efficiency of a radical organic light-emitting diode.Organic  Organic π-conjugated radicals have recently joined the ranks of high-efficiency light-emitting materials, however, their light-emission mechanism is still a matter of debate. Here, the authors highlight a recently proposed luminescent enhancement mechanism and record-breaking efficiency of a radical organic light-emitting diode. Under electric bias, holes and electrons are injected into OLEDs from the anode and cathode. The injected carriers recombine on light-emitting molecules to generate excitons followed by light emission. The luminous efficiency of the OLEDs significantly depends on the type of light emitter used. In the closed-shell light-emitting molecule system 3 and thermally activated delayed fluorescence4, resulting in high-efficiency OLEDs with an EQE of over 30%5. On the other hand, recently, open-shell neutral organic π-conjugated radicals (OPRs) have received great attention as a new class of highly efficient light-emitters for OLEDs  and the lowest-lying excited state (D1) are doublets, and the D0–D1 electronic transition is spin-allowed, making all the generated excitons bright. Therefore, the IQE can reach 100% when OPRs are used as light emitters, as shown in Fig. OLEDs are devices that convert electricity into light and are used as displays and in lightingtem Fig. , the groEDs Fig. 6–10. In 12. One is the delocalization of the lone pair to the π-conjugated system to form OPRs. One representative example is the triphenylmethane (TPM) radical. The lone pair delocalizes to the three phenyl rings and becomes stabilized by π-conjugation. The other is the introduction of steric hindrance near the radical to prevent the chemical reaction. One representative example is the trismethane (TTM) radical. The substituted chlorine atoms on the phenyl rings act as steric hindrance, and thus, it is more difficult for other molecules to be close to the radical than the unsubstituted TPM radical, bestowing further stabilization to the TTM radical. The TTM radical is stable at room temperature in contact with air and has light-emitting properties, albeit at a low PLQE of several percent. However, the TTM radical is weak against photoexcitation and immediately undergoes decomposition. The recent development of OPRs has overcome these difficulties. The recently developed OPRs are summarized in Fig. 13. The TTM-1Cz-based OLED showed deep red electroluminescence with a peak wavelength of 700 nm. However, the achieved maximum EQE was limited to 2.4%. The breakthrough of efficiency improvements was brought by Ai et al.7 in 2018. They incorporated 3-substituted-9-phenyl-9H-carbazole (3PCz) and 3-substituted-9--9H-carbazole (3NCz) into the core TTM radical. TTM-3PCz and TTM-3NCz doped in solid 4,4-bis(carbazol-9-yl)biphenyl (CBP) films (3.0 wt%) exhibited high PLQEs of 60.4% at 695 nm and 85.6% at 707 nm, respectively. TTM-3NCz showed a much higher resistance than TTM in cyclohexane solution under photoexcitation. The PL half-life of TTM was ~20 s, while the PL intensity of TTM-3NCz did not change after several thousand seconds. In addition, TTM-3NCz and TTM-3PCz exhibited high thermal resistance, enabling OLED fabrication using a thermal evaporation process. OLEDs were fabricated using these radicals as light emitters. Surprisingly, a maximum EQE of 17% for TTM-3PCz and 27% for TTM-3NCz were achieved. This high EQE of 27% is close to the theoretical limit of 30%. Thus, Ai et al. proved that the IQE can reach 100% when using radical emitters7. This success was achieved by improvements in radical stability and PLQE. The spread of π-conjugation in the substituted TTM is greater than that of TTM, and the lone pair can delocalize more, which is the reason for the stability improvement. In the luminous efficiency, in general, a large overlap between the singly occupied molecular orbital located at the core TTM and the highest occupied molecular orbital located at the carbazole substituent is necessary for a large oscillation strength. In addition, Abdurahman et al. proposed the PLQE enhancement mechanism by intensity borrowing from an intense high-lying electronic transition to a weak low-lying charge-transfer electronic transition9. However, the mechanism of luminous efficiency improvement is still a matter of debate.Organic carbon radicals have lone pairs on carbon atoms and are generally known as unstable chemical species. The insufficient electronegativity of the carbon nucleus cannot bind the lone pair to the carbon atom, and the loosely bound lone pair easily reacts with other chemical species. There are two strategies to stabilize organic carbon radicals Fig. 11,12. On1TTM, M2TTM, and M3TTM), and M3TTM, having the highest symmetry, showed the highest PLQE10, which is not explained by the above two mechanisms , resulting in an increase in the PLQE to 93% in a film doped with a CBP host. The OLED using M2TTM-3PCz was fabricated and showed a record-breaking EQE of 28% at a wavelength of 689 nm. However, efficiency roll-off was observed in the low current density region, and the efficiency decreased at high luminance. In the doped film, time transient PL measurements were performed, and the formation of the intermolecular charge-transfer complex between the luminescent radicals and CBP host was observed, resulting in long-lived excited states on the order of microseconds. In closed-shell systems, TTA, a long-lived triplet-to-triplet reaction, is thought to be responsible for the efficiency roll-off at high current densities14. Even in the open-shell system of this study, reactions between doublet and doublet excitons can occur and cause roll-off. The interaction between radicals and host molecules will need to be studied in detail to avoid generating long-lived intermolecular CT states. They also synthesized a main-chain copolymer of mesitylated TTM and 9,9-dioctyl-9H-fluorene (PFMTTM) based on the postpolymerization radicalization of a nonradicalized precursor, which is the first report of a luminescent polymer with radicals embedded in the main-chain. PFMTTM showed significantly redshifted emission beyond 800 nm, which is expected to be a new near-infrared light source (Fig. Very recently, Murto et al. reported the synthesis of mesityl group-substituted TTM radicals (Msms Fig. . They exost Fig. . This prrce Fig. .Fig. 2OrWhile the present postpolymerization radicalization strategy did not convert all the parent units to their radical forms, as evident from the luminescence from nonradicalized units Fig. , alterna


Canine mammary tumors are the most common tumor disease of female dogs, and adjuvant chemotherapy often results in multi-drug resistance. Currently, the mechanisms underlying the development of tumor multi-drug resistance are unclear. The translation of research applications that can be used to effectively overcome tumor resistance is similarly hampered. Therefore, it is urgent to construct multi-drug resistance models of canine mammary tumors that can be used for research, to explore the mechanisms and means of overcoming resistance.In this study, the canine triple negative breast cancer cell line CMT-7364 was induced to develop multidrug resistance using doxorubicin by high-dose drug pulse method. The drug resistance and the expression of drug transport pumps of the cells was verified by CCK8 assay, immunoblotting, qPCR and immunofluorescence. Next, we used scratch assay and Transwell invasion assay to compare the migration and invasion abilities of the two cell lines and examined the expression of EMT-related proteins in both using immunoblotting. The differences of transcriptome between parental and drug-resistant cell lines were detected by RNA-seq sequencing. Finally, mouse xenograft models of drug-resistant and parental cell lines were constructed to evaluate the tumorigenic ability.After more than 50 generations of continuous passages stimulated by high-dose drug pulse method, the morphology of drug-resistant cell line CMT-7364/R tended to be mesenchymal-like and heterogeneous under light microscopy compared with the parental cell line CMT-7364/S, and developed resistance to doxorubicin and other commonly used chemotherapeutic drugs. In CMT-7364/R, BCRP was expressed at higher levels at both transcriptional and protein levels, while P-glycoprotein was not significantly different. Secondly, the migration and invasion ability of CMT-7364/R was significantly enhanced, with decreased expression of E-cadherin and increased expression of vimentin and mucin 1-N terminus. Finally, mouse xenograft models were constructed, while there was no significant difference in the volume of masses formed at 21 days.In summary, by using the canine mammary tumor cell line CMT-7364/S as the parental cell line, we successfully constructed a multidrug-resistant CMT-7364/R with high-dose drug pulse methods. Compared to its parental cell line, CMT-7364/R has decreased growth rate, overexpression of BCRP and increased migration and invasion ability due to EMT. The results of this study showed that CMT-7364/R might serve as a model for future studies on tumor drug resistance. Canine mammary tumors (CMTs) are the most common tumors in female dogs, accounting for 50–70% of all tumors. Of these, canine mammary cancer (CMC) accounts for 50% of CMTs –3. CMTs Although the utility of chemotherapy has not been confirmed by large-scale studies, chemotherapy remains the best adjuvant option after surgical resection due to the highly heterogeneous and aggressive nature of CMTs. Unfortunately, however, tumor multi-drug resistance often causes chemotherapy failure . Therefoin vitro development has a long history and can be used to study the mechanisms of chemoresistance to chemotherapeutic agents. The primary method of constructing tumor-resistant cell lines in the laboratory is to repeatedly expose tumor cells to drugs during cell culture. Cells that survive this “survival stress” are screened for greater drug tolerance than their parental cells. The induction of drug resistance in cell lines in vitro often takes 3–18 months, and many factors are involved, including the choice of parental cell line, the type of drug used, the dose of the drug, and the method and interval of administration  canine mammary tumor cell lines is derived from a clinically spontaneous canine breast tumor. The case was a 13-year-old female dog with a histopathological diagnosis of grade III ductal papillary and triple negative mammary carcinoma. After primary isolation and culture, a stable passaged cell line has been formed and cultured in DMEM medium containing 10% FBS  and 1% penicillin-streptomycin in a culture environment with a temperature of 37°C and a COer study . were gr50 value. The drug was allowed to act for a period of 48 h. In a culture environment with a temperature of 37°C and a CO2 content of 5%, the drug-containing culture solution was discarded, then washed twice using PBS and add normal culture medium. Thereafter, liquid is changed every 1–2 days to remove metabolic waste and floating dead cells. After the surviving cells regain their ability to grow, they are passed on and the drug pulse is given again according to the above method. This cycle is repeated for at least 50 generations. We named the final cell line obtained in the normal passaged group as CMT-7364/S and the cell line in the high-dose drug pulse group as CMT-7364/R.We selected CMT-7364 as the parental cell line and set up a normal passaged group and a high-dose drug pulse group. When developing the drug-resistant cell line, a high-dose drug pulse method is used. After reaching the logarithmic growth stage, cells were subjected to a drug pulse by adding doxorubicin to the medium at concentrations up to the IC3 and 1 × 104 cells per well for cell growth Assay and cell viability assay respectively. Then the cells were incubated for 24 h to allow attachment. For cell growth assay, cells in triplicate were incubated with 10 μL volume Cell Counting Kit-8 for 2 h and then detected in microplate reader  with a 450 nm filter at every 24-h interval for 7 days. When cell growth curve was plotted, the cell growth doubling time was calculated with Graphpad Prism 9. For cell viability assay, then different concentration of drugs were mixed with cells. After 48 h, a 10 μL volume of CCK8 was added to each well according to the manufacturer's insctructions, measuring the optical density (OD) in the same at 450 nm, and the IC50 was calculated using Graphpad Prism 9. Subsequently the resistance index was calculated according to the following equation:CMT-7364/S and CMT-7364/R cell lines were seeded in triplicate in 96-well plates with 5 × 105 cells per well and three parallel wells marked with a straight line at the bottom of each well. After the cells formed a monolayer and fully adhered to the bottom of the well, the supernatant was discarded, and a sterile 200 μL pipette tip was used to scratch the adherent cells along the marked line in a single direction, creating a linear region of detached cells, followed by washing the detached cells with sterile PBS. In this way, a regular and uniformly wide blank area was created in each well, and then 2 mL of serum-free DMEM medium was added. At this point, the width of the scratch was photographed as 0 h, and then photographs were taken at 24 h and 48 h in the same area. The images were processed using Image J software to calculate the scratch area at different time points for each group. For Transwell invasion assay, we used 8.0 μm Transwell Permeable Supports (Corning). The upper chamber was pre-coated with Matrigel Matrix (BD Biosciences), and 600 μL medium containing 10% FBS was added to the lower chamber. The cell was plated at a density of 5 × 104 using 200 μL serum-free DMEM in the upper chamber. After incubation for 24 h, cells that did not invaded through the membrane were mechanically removed with a cotton swab. Next, 4% paraformaldehyde was used to fix the cells on the bottom surface of the membrane for 10 min, and then cells were stained with a 0.4% crystal violet solution. The invaded cells were imaged and quantified under microscopy.Two types of cells were seeded into a 6-well plate, each with 5 × 105 cells/well and incubated at constant temperature for 24h to allow adequate wall attachment.The cells were treated with 5 and 10 μM doxorubicin for 24 h, respectively. After that, the cells were extracted and preserved in 70% ethanol at 4°C for 2 h. The cells were then centrifuged at 1,300 rpm for 5 min to remove the ethanol before being rinsed with PBS and centrifuged again. Using a BD FACSCalibur flow cytometer with selective gating to remove doublets of cells, the cells were stained with propidium iodide (PI) to form PI-DNA complexes in order to assess cell cycle distribution. Data were analyzed using FlowJo V10 software.Flow cytometry was used to determine the cell cycle distribution of chemotherapeutic-treated cells. CMT-7364/S and CMT-7364/R cell lines at logarithmic growth stage were inoculated in 12-well plates at 2.5 × 106 cells per well. After fully attachment, protein extraction was performed with ice-cold lysis buffer  following the manufacturer instructions, and then proteins were quantified using the BCA protein assay kit . Equal protein amount (20 μg protein per lane) were seperated on a 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membranes . The membranes were blocked with 5% skim milk diluted with TBST for 1 h at room temperature, followed by incubation with primary antibody overnight. Primary bodies included anti-P-gp , anti-GAPDH , anti-E-Cadherin , anti-Vimentin , anti-MUC1-N , anti-BCRP . Subsequently, membranes were incubated in horseradish peroxidase (HRP)-conjugated secondary antibodies, anti-rabbit IgG  for 1 h at 37°C Finally, using a chemiluminescence imaging analysis system  to detect signals. The quantification of the bands was done by Image J software.Both type of cells, CMT-7364/S and CMT-7364/R were plated in a 6-well plate at 1 × 10mdr1, abcg2 and gapdh were provided in Additional file. All the real-time PCR reactions were performed using SYBR Premix Ex II (Tli RNaseH Plus) in Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems). The Ct for quantification and fold change for target genes was normalized by internal control.Total RNA was extracted using TRIzol Up (TransGen Biotech) following the manufacturer's protocol. RNA concentrations were measured using a NanoDrop (NanoDrop Technologies). RNA was reverse transcribed to cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). The gene GAPDH was used as an internal control. The primers used for 5 per well and incubated at 37°C in a humidified atmosphere containing 5% CO2. After the cells were fully adhered to the coverslips, the medium was discarded and 1 mL of 5 μM DOX was added to each well and incubated for 12 h, then washed the cells with PBS three times. The cells were fixed with 4% paraformaldehyde for 20 min followed by another three washes with PBS at room temperature. After that, 10 μL of antifade mounting medium with DAPI  was added dropwise to each coverslips for sealing. The coverslips were examined by confocal microscopy as soon as possible.Attach round glass coverslips pretreated with TC  to the bottom of the wells of a 12-well plate. Both type of cells were seeded on the glass coverslips at a density of 1 × 105 cells per well) were plated on coverslips. After 24 h, the cells were fixed in 4% paraformaldehyde solution for 20 min at room temperature, and permeabilized in 0.5% Triton X-100 for 5 min. Then, the cells were washed three times, incubated with anti-P-gp or anti-BCRP(1:500) overnight at 4°C, then incubated with appropriate conjugated secondary antibodies for 1 h at 37°C. After that, 10 μL of antifade mounting medium with DAPI was added dropwise to each coverslips for sealing. The coverslips were examined by confocal microscopy as soon as possible.Cells (1 × 106 cells per well). After 24 h of incubation, cells were collected by trypsinisation, snap-frozen on dry ice and stored at –70°C for RNA extraction. The TransZol Up was used to extract RNA from the cells. Total RNA was purified from animal cells using EasyPure RNA Kit , according to the manufacturer's protocol. RNA-seq data analyses were performed by DIATRE Biotechnology, Shanghai, China. All datasets generated and analyzed within this study are available in the NCBI's Sequence Read Archive by accession number PRJNA914497.Cells were seeded in triplicate into 6-well plates (1 × 106 cells were resuspended in 200 μL PBS. The mice were monitored weekly for the growth of tumors. Tumor growth (tumor length and width) and body weights were measured every 3 days until the study was terminated on day 21. Tumor volume was calculated using formula: length x width2/2. At the end of the experiment, all the mice were first anesthetized with isofluorane and then euthanized via CO2 asphyxiation to collect xenograft tumors and major organs. All animal procedures were approved by the Institutional Animal Care and Use Committee of China Agricultural University.Tumor xenografts were established in 5-week-old BALB/c nude mice  by subcutaneous injection of cells into the mammary fat pad. There were three mice in each group. For each tumor, 5 × 10t-test. Data are shown as mean ± SEM. The p < 0.05 were deemed statistically significant.Statistical analysis was performed and graphs were generated with GraphPad Prism 9 or SPSS. Statistical differences in this study were explored by Student's 50 of 63.20 ± 1.350 μM compared to its parental cell line with an IC50 of 8.780 ± 0.157 μM and a resistance index greater than 5 (RI = 7.198), which meets the requirements for a drug-resistant cell line (in vitro developed drug-resistant canine mammary tumor cell line as CMT-7364/R (Resistant) and the corresponding parental cell line as CMT-7364/S (Sensitive). After more than 50 consecutive generations of high-dose drug shock induction, obvious changes have appeared in cell morphology. CMT-7364/S is mostly shuttle-shaped, with clear boundaries, uniform size, and strong refractivity, while CMT-7364/R is of heteromorphism with weak refractivity . It indicates that the proliferative capacity of CMT-7364/R was diminished compared to CMT-7364/S.After 50 consecutive generations of passages according to the method described in Section 2.2, we examined the sensitivity of the cells to DOX by calculating the relative survival rate of the cells. The drug-resistant cell line was less sensitive to DOX with an ICell line  9). In . In 50 oactivity . During p < 0.01 and 0.0001, respectively). This indicates that CMT-7364/R is more resistant to the cell cycle blocking effect of DOX.Doxorubicin, a cycle non-specific anticancer chemotherapeutic agent, acts on cells of all phases and can cause G2/M phase cell cycle arrest. Therefore, to fully verify the resistance of CMT-7364/R to DOX, we examined the cell cycle distribution of both cell lines and the effect of DOX on them. The percentage of cells in each phase in different groups and their distribution are shown in Furthermore, doxorubicin is capable of autofluorescence after excitation with excitation light and is a commonly used model drug. Therefore, we observed the uptake of doxorubicin by CMT-7364/R and its parental cell lines after 12 h. The results are shown in 50 of several other drugs commonly used clinically for chemotherapy of canine mammary tumors on parental and resistant cell lines, including 5-fluorouracil (5-FU), paclitaxel (PTX), and cisplatin (DDP), and the results of their IC50 are shown in To verify whether CMT-7364/R is multi-drug resistant, we also examined the IC****p < 0.0001). In addition, we examined the differences in transcript levels of these two genes by RT-qPCR, and the results are shown in the ****p < 0.0001). To more visually verify the expression of P-gp with BCRP, we performed immunofluorescence assay and observed the expression of P-gp and BCRP among the cells using confocal microscopy, as shown in There are numerous mechanisms by which tumor cells develop multi-drug resistance, and one of the most common mechanisms is the presence of over-expression of ABC transporter pumps. To investigate the mechanisms by which CMT-7364/R develops multi-drug resistance, we examined two common ABC transporter proteins, including P-gp and BCRP. No significant difference was seen in the expression level of P-gp in CMT-7364/R compared to its parental cell line, while the expression level of BCRP was significantly elevated . By semi**p < 0.01). This result indicated that the migration ability of CMT-7364/R was enhanced compared to CMT-7364/S (*p < 0.05). This result indicated that the invasion ability of CMT-7364/R was enhanced compared to CMT-7364/S.Given the mesenchymal changes in the morphology of the cells, we hypothesized that the migration and invasive ability of the cells might be altered. Firstly, the results of the cell scratch healing assay showed that the difference in the area of scratch healing due to cell migration was statistically significant in CMT-7364/R compared to the parental cell line aft. After 48 h, the percentage of scratch healing area in the CMT-7364/R group was 82.30 ± 1.054 compared with 73.30 ± 1.600 in the CMT-7364/S group (T-7364/S . SecondlT-7364/S . After 2****p < 0.0001), which are consistent with the expression profile of the cell line in which the EMT process occurred. Mucin 1 (MUC1) is a transmembrane protein of the mucin family, consisting of an extracellular N-terminal and an intracellular C-terminal, which can form heterodimers; the longer N-terminal peptide acts as a physical barrier and is significantly correlated with cell adhesion, migration, and invasion. As we mentioned above the difference in adhesion ability between the two cell lines, we examined the expression level of MUC1-N. As a result, the expression level of MUC1-N was significantly elevated in CMT-7364/R  during the process of high-dose drug pulse development. For this purpose, we examined the expression of the relevant proteins, and the results are shown in T-7364/R .To investigate the potential mechanism of the development of drug-resistance in CMT-7364/R, we analyzed transcriptome expression between drug-resistant cells and parental cells by RNA-sequencing. GO analysis showed a significant improvement in the cell adhesion capacity of CMT-7364/R, which is consistent with the phenomenon we observed in cell culture. According to the results of KEGG analysis, the MAPK pathway of CMT-7364/R is activated .In this study, xenograft models of both drug-resistant cell lines and parental cell lines were constructed. After inoculating tumor cells for 5 days, gradually enlarging solid mass with a clear outline in the mammary area on the left side of the groin can be seen in mice injected with either the drug-resistant cell line or the parental cell line. However, the masses that grow in mice inoculated with CMT-7364/R are larger in size and often ulcerate. Within 21 days, the volume of the mass was recorded every 3 days. After 21 days, the mice were sacrificed, and the final volume of the mass is measured . Masses in vitro drug-resistant cell models have been established for several kinds of tumors by means of in vitro construction, including breast cancer, liver cancer, colon cancer, and melanoma, among others  transporter protein, which uses ATP to pump chemotherapeutic drugs out of the cell, is a common mechanism of resistance, and overexpression of P-gp and BCRP is most frequently seen in canine breast cancers that develop multi-drug resistance. Completely unraveling this network remains a major challenge and is essential for identifying new therapeutic targets. However, in order to understand the mechanisms involved, stable cell models are urgently needed. In human medicine, g others . In contin vitro development, CMT-7364 initially showed massive cell population death after drug stimulation, and large areas of cell de-adhesion were visible microscopically, floating in the culture medium. Very few cell populations remained adherent and proliferative after elution. After several months of induction by continuous high-dose drug pulse method, CMT-7364 gradually developed resistance to the drug and could still adhere to the bottom of the dish after receiving the same dose of drug. Under the optical microscope, the cells gradually developed from a shuttle shape with clear borders initially to a morphology with unclear borders and a strong anisotropic phenotype. By the CCK-8 method, we confirmed that the cells were multi-drug resistant and named the obtained resistant cell line as CMT-7364/R and the parental cell line as CMT-7364/S.Thus, this study developed a canine mammary tumor drug-resistant cell line by high-dose drug pulse method. During We first compared the proliferative capacity of the two cell lines, and by comparing the PDT of both, we found that the proliferative capacity of CMT-7364/R was significantly reduced. In addition, doxorubicin has a strong antitumor effect as a cycle non-specific anticancer chemotherapeutic agent that causes G2/M phase block of the cell cycle . And by Secondly, since overexpression of the ABC transporter pump is one of the common mechanisms mediating multi-drug resistance in tumors, we examined the expression of P-gp, which plays a major role in breast cancer together with BCRP, the most well-characterized drug transporter in the ABC protein superfamily, encoded by the ABCB1 (formerly known as MDR1). Among human medical oncology cases, P-gp is probably best known for its role in mediating multi-drug resistance to chemotherapeutic drugs. In recent years, tumor resistance has begun to gain attention in the veterinary field due to a aging trend in the average age of companion animals and an increase in tumor incidence , 18. Curin vitro. EMT is an important process in normal embryonic development and is the most common cause of tumor invasion and metastasis initiation causes  during induction by high-dose drug pulse n causes . Typicaln causes , for whin causes . The resn causes . It was in vitro canine breast cancer cell lines and naturally occurring canine mammary tumors has proven to be a valuable tool for advancing our understanding of animal cancer and can also serve as excellent translational models for human breast cancer  can be found in the article/supplementary material.The animal study was reviewed and approved by the Animal Ethics Committee of China Agricultural University.CZ and DZ designed the whole study. CZ performed the experimental work and data analysis and wrote the manuscript. ZL, XL, and PS took part in some of the experiments. ZL and DZ revised the manuscript. All authors read and approved the manuscript.


Finally, one of the studies was eliminated because of unanalyzable results. All studies reached a high methodological quality. A total of 289 patients were studied with follow-up periods from 2 weeks to 1 year. Statistical significance was only found, with respect to the use of adjunctive metronidazole, in the pooled analysis of the studies (p = 0.02) and in the analysis of the radiographic values reported on peri-implant marginal bone levels, in the studies with a 3-month follow-up (p = 0.03). Discrepancies in the use of systemic metronidazole require long-term randomized clinical trials (RCTs) to determine the role of antibiotics in the treatment of peri-implantitis.Peri-implant diseases are pathological conditions that affect the survival of dental implants. Etiological studies are limited, accepting a prevalence of 20% at the implant level and 24% at the patient level. The benefits of adjuvant metronidazole are controversial. A systematic review and meta-analysis of RCTs according to PRISMA and PICOS was performed with an electronic search over the last 10 years in MEDLINE (PubMed), WOS, Embase, and Cochrane Library. The risk of bias was measured using the Cochrane Risk of Bias tool and the methodological quality using the Jadad scale. Meta-analysis was performed with RevMan version 5.4.1, based on mean difference and standard deviation, with 95% confidence intervals; the random-effects model was selected, and the threshold for statistical significance was defined as  For cliPorphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Eikenella corrodens, Filifactor alocis, Aggregatibacter actinomycetemcomitans, and Staphylococcus aureus; however, there is controversy about the homogeneity of biofilm composition in both pathologies  statement   format: In patients with peri-implantitis, is adjuvant local or systemic treatment with metronidazole effective on changing signs of inflammation and bone destruction?(P) Population: patients receiving adjuvant treatment with local or systemic metronidazole.(I) Intervention: surgical or non-surgical treatment of peri-implantitis.(C) Comparison: patients not receiving adjuvant treatment with metronidazole.(O) Outcomes: primary outcomes: bleeding on probing, bleeding rate, suppuration, and probing depth. Secondary outcomes: clinical attachment level, bone level (radiographic test), and implant failure.(S) Study design: randomized controlled trials.2.2Clinical Implant Dentistry and Related Research, Clinical Oral Implants Research, International Journal of Oral and Maxillofacial Implants, Journal of Clinical Periodontology, and Journal of Periodontology.Four electronic databases were searched for relevant articles published in the last 10 years up to December 2022: MEDLINE (through PubMed), WOS, Embase, and Cochrane Library. The search filter “Randomized Controlled Trial” was applied. The electronic search was complemented with a manual search in the following journals: The following Medical Subject Headings (MeSH) terms were used: “Dental implants,” “Dental plaque,” “Peri-Implantitis/prevention and control,” “Anti-Bacterial Agents/therapeutic use,” “Metronidazole/therapeutic use,” and “Humans.” Boolean AND/OR operators were used to refine the search. In addition, relevant studies from the grey literature and reference lists of the included studies (cross-references) were also examined. The search strategy and PICOS format are shown in 2.3The inclusion criteria were as follows:a) RCTs (single or double-blind) performed in patients with peri-implantitis defined as bleeding, bone loss ≥2 mm, and/or suppuration on peri-implant probing (≥ 4 mm)vs. single surgical or non-surgical treatmentb) Studies comparing the efficacy of local/systemic metronidazole adjuvant therapy c) Articles published in EnglishThe exclusion criteria were as follows:a) Less than five patients per treatment groupb) Lack of clinical or radiographic data on bone destructionc) Case series or clinical casesd) Undefined cases and non-relevant studies2.4κ)  independently selected titles and abstracts. Cohen’s kappa index (κ)  was calcSubsequently, the selected articles were obtained for review, data extraction, and inclusion. Bibliographic references of the included studies were also reviewed as possible sources of additional studies.2.5Two reviewers (NL-V and AL-V) independently assessed the quality of each RCT according to the Cochrane Risk of Bias tool (RoB2) . Five do2.6The Jadad scale   defines 2.7I2 when few studies are used in the meta-analysis due to the expected methodological heterogeneity in the included studies. The heterogeneity was interpreted as follows: low I2 = 25%, moderate I2 = 50%, and high I2 = 75%. The threshold for statistical significance was defined as p < 0.05.Meta-analysis was performed using Review Manager . A meta-analysis was performed based on the mean difference (MD) and standard deviation (SD) to estimate effect size, with 95% confidence intervals (CI) for adverse event outcomes. The random-effects model was selected considering the uncertainty in 33.1κ = 80% , with the study by 3.3Of the five included studies , only foAll studies met the domains of random sequence generation and blinding of participants and personnel; one study was biased in the incomplete outcome domain , and all3.4The selected studies included a total of 289 patients, and follow-up periods ranged from 2 weeks to 1 year. The studies with the most complete follow-up were those by 3.5Only four of the included studies were used for meta-analysis . The higI2 = 45%) (I2 = 0%)  (p = 0.02) (95% CI) (p = 0.69).Heterogeneity was moderate in the analysis of grouped studies I2 = 45% I2 = 45% I2 = 45% I2 = 45% I2 = 45% I2 = 45% (I2 < 75%). Statistical significance (p = 0.03) was found only in the 3-month follow-up subgroup. The bone level loss was also analyzed separately in studies using systemic antibiotics (I2 = 0%), so a fixed-effects model was performed; however, no statistical significance was found between groups (p = 0.60) , so this systematic review aimed to evaluate the efficacy of metronidazole as an adjuvant in the treatment of peri-implantitis. In total, five RCTs were included in the analysis, of which four were analyzed and one was eliminated for providing non-analyzable data.The absence of BoP is evidence of the absence of inflammation, which should be the first target in the treatment of peri-implantitis, and according to recent recommendations, the results of peri-implantitis treatment should be evaluated after 6 months of healing, based on outcomes including multiple parameters: bone fill, peri-implant soft tissue recession, BoP, SoP, and PPD . HoweverMombelli and Decaillet  highlighp = 0.02). A statistically significant result (p = 0.03) was also obtained in the analysis of studies with shorter follow-up (3 months).According to the results obtained in our study, the pooled RCT analysis demonstrated the effect of metronidazole as an adjuvant treatment after 12 weeks (vs. 1.02 mm) and CAL (2.14 vs. 0.53 mm) in the test group compared with the control group. Similarly, Shibili et al . In contrast, Shibli et al., with metronidazole and amoxicillin and the same follow-up time, found no significant differences between the groups (A. actinomycetemcomitans, as this germ is known to resist mechanical treatment particularly well (A. actinomycetemcomitans and 41 negative cases, also found no specific benefit from the use of metronidazole in combination with amoxicillin.li et al . with mereatment . In contreatment . found ne groups . In thisrly well ; howeverCertain similarities in terms of biomarkers of bone destruction and causative microbial agents, in peri-implant and periodontal pathologies and the frequent coincidence of both in the same patient, have led some researchers to investigate both pathologies together or to design studies on peri-implantitis based on those performed on periodontitis , althougOn the other hand, it is known that an individual’s immune response to bacterial aggression is influenced by genetic and epigenetic factors, as well as environmental factors. It has been shown that inflammatory diseases, such as peri-implantitis and periodontitis, can be enhanced by epigenetic modifications in certain subjects . A recenThe present meta-analysis has some limits that must be emphasized. On the one hand, the included studies used local and systemic adjuvant antibiotics; on the other hand, only the study by On the other hand, The outcome reports of the included studies were conducted at different time periods, and although Sanz et al . recommeFinally, although all studies used radiographic evaluation by intraoral radiography to assess the peri-implant bone loss, there are other useful radiographic techniques, such as multislice computed tomography and cone-beam volumetric imaging, which offer certain advantages to implant dentistry, such as the representation of intraosseous lesions in three planes, on a real scale and without overlapping or distortion. In addition, computer-assisted image analysis, such as subtraction radiography, allows the detection of small changes in bone density . The difAll this could bias the results, and therefore, the results obtained in our meta-analysis should be taken with caution.Therefore, we consider that the decision on a specific adjuvant antibiotic treatment should be made individually for each patient, taking into account the severity and extent of peri-implantitis and possible concomitant periodontitis, as well as the number of affected areas requiring treatment. Likewise, the potential risk of the development of strains resistant to metronidazole or to the chosen antimicrobial combination should be taken into account, and the protocol to be followed in each indicated situation should be carefully decided . On the 5Adjuvant local or systemic administration of metronidazole in the treatment of peri-implantitis remains of questionable efficacy and with serious discrepancies among investigators.Due to the heterogeneity of the types of treatment, the reported administration protocols, and, ultimately, the heterogeneity of the studies, we have not been able to draw definitive conclusions about its effect in the adjuvant treatment of peri-implantitis.In certain pathologic situations of peri-implantitis, metronidazole, alone or in combination with other antibiotics, could be beneficial as an adjuvant to surgical treatment, always weighing the benefits against the disadvantages.Long-term RCTs with standardized methodologies would be desirable and justifiable to determine the exact role of metronidazole as an adjuvant treatment for peri-implantitis.Conceptualization: NL-V and AL-V. Methodology: NL-V. Validation: JB-R. Formal analysis: NL-V. Resources: AL-V. Data curation: NL-V. Writing—original draft preparation: NL-V. Writing—review and editing: NL-V and AL-V. All authors contributed to the article and approved the submitted version.


Physical activity (PA) after metabolic and bariatric surgery (MBS) can influence weight loss, health status, and quality of life. Known mediators to participate in PA are enjoyment, self-efficacy, and social support. Little is known about PA behavior in MBS individuals. The aim of this study was to explore levels of PA and the PA mediators enjoyment, self-efficacy, and social support before and after MBS and to investigate changes over time.Adults scheduled to undergo MBS were recruited from a Swedish university hospital. Accelerometer-measured and self-reported PA, body weight, and PA mediators were collected at baseline and at 12 to 18 months post-surgery.p < 0.05) and from 270.5 to 303.5 min/day (p < 0.01), respectively. Step counts increased significantly from 6013 to 7460 steps/day (p < 0.01). There was a significant increase in self-reported PA, enjoyment, self-efficacy for exercise, and positive social support from family. The increase in PA mediators did not lead to a significant change in time spent in moderate to vigorous PA.Among 90 individuals included, 50 completed the follow-up assessment and had valid accelerometer data. Sedentary time (minutes/day) was unchanged, but sedentary time as percentage of wear time decreased significantly from 67.2% to 64.5% (p<0.05). Time spent in light PA and total PA increased significantly from 259.3 to 288.7 min/day (The increase in PA-mediators was not associated with an increase in moderate to vigorous PA, but the strengthened PA mediators suggest potential for an increase in moderate to vigorous PA in patients undergoing MBS.The online version contains supplementary material available at 10.1007/s11695-023-06887-7. Persons seeking metabolic and bariatric surgery (MBS) tend to spend most of their time in sedentary behaviors and perform low levels of physical activity (PA) , 2. PartPA after MBS is important because it contributes to greater weight loss, helps maintain a lower weight, and can reduce risk of diseases and improve quality of life –10. WorlWeight loss after MBS can facilitate participation in PA; however, the literature on PA after MBS is conflicting. A meta-analysis from 2019 found no increase in objectively measured PA within 6 months post-surgery but found significant improvement after 6 months , while oPsychological factors are central to the decision to perform PA; the main PA mediators are enjoyment, self-efficacy, and social support. Self-efficacy is a person’s confidence in their ability to perform a particular behavior . Weight The study included adults ≥ 18 years who were scheduled to undergo primary Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy (SG) at a Swedish university hospital. Participants were recruited between June 2015 and February 2018. The indication for MBS was based on a modified version of the 1991 National Institutes of Health criteria, including body mass index (BMI) ≥ 35 with or without obesity-related comorbidity, previous weight loss attempts, mental stability, and no substance abuse ≥ 2 years before surgery . In accoMeasures of PA, self-efficacy for exercise, PA enjoyment, social support for exercise, and body weight were collected prior to surgery and at the1-year follow-up visit which, due to medical and practical reasons, took place at 12 to 18 months post-surgery. This is an appropriate time to evaluate lifestyle changes after a significant weight loss. Medical data and education level were retrieved from the Scandinavian Obesity Surgery Registry, a national quality and research registry .PA was measured using an accelerometer . The accelerometer was sent to participants by mail, with instructions to wear it on an elastic belt on the back or hip during all waking hours for 7 consecutive days, except during showering and bathing. Participants were asked to keep an exercise diary, reporting when the accelerometer was put on and taken off, and type of PA performed. Data was reintegrated to 60-sec epochs  and analyzed using ActiLife6 . Valid wear time was defined as ≥ 10 h/day for ≥ 3 days . Counts The Swedish National Board of Health and Welfare’s validated questions about the frequency of vigorous and moderate PA were used to measure self-reported PA . The twoEnjoyment was measured using the validated Physical Activity Enjoyment Scale (PACES)  modifiedSelf-efficacy was assessed using the validated Self-Efficacy for Exercise Scale (SEES) , which mSocial support was assessed using the validated 13-item Social Support for Exercise Scale (SSES) , which ip = 0.05. Categorical data was tested with the Chi-2 test. Means and standard deviations (SDs) were calculated. Normal distribution was evaluated graphically using histograms. Paired samples t tests were used to test for changes in PA and PA mediators over time. Effect size of within-group change was estimated by calculating the standardized response mean (SRM), i.e., the mean change divided by the SD of change. The SRM was evaluated according to standard criteria: < 0.20 = trivial, 0.20–0.49 = small, 0.50–0.79 = moderate, and ≥ 0.80 = large [p-value <0.05 was considered statistically significant. Analysis was performed using SPSS version 27 .Calculations based on a study assessing PA in women undergoing RYGB  indicate = large . A p-valA total of 147 individuals provided consent, but 57 did not complete the baseline assessments . The change in mean sedentary time (min/day) was not significant (p = 0.79); however, sedentary time as percentage of wear time decreased significantly from 67.2% pre-surgery to 64.5% post-surgery (p < 0.05). Participants increased their time spent in LPA from 259.3 to 288.7 min/day (p < 0.05). Moderate to vigorous PA was 11.2 min/day pre-surgery and 14.9 min/day post-surgery (p = 0.06). Total PA increased from 270.5 to 303.5 min/day (p < 0.01). Altogether, about 30 min/day of non-wear time was replaced with about 30 min/day of LPA. Steps increased from 6013 steps/day pre-surgery to 7460 steps/day post-surgery (p < 0.01). The summary score for self-reported PA increased from 7.9 to 10.5 (p < 0.001). Almost one-fourth of participants increased their MVPA by > 10 min/day post-surgery (Table Table p < 0.001). Self-efficacy for exercise increased from 4.2 to 5.3 (p < 0.01). Positive social support from family for PA increased from 20.1 to 22.7 (p < 0.05). Negative social support from family and positive social support from friends remained unchanged  remained unchanged.The PA mediator enjoyment, self-efficacy for exercise, and positive social support from family increased significantly post-surgery, in line with previous studies , 15, 32.The increase in mediators for PA indicates a positive change in the participants’ perception of PA, which can be a first step towards a change in behaviors. However, it did not lead to a significant change in MVPA at group level, which has also been shown in previous studies , 14. To The proportion of individuals meeting PA recommendations in a population may vary. A Swedish cohort study observed fivefold higher odds for high sedentary time and low MVPA in persons with BMI ≥ 35 compared to persons with BMI < 24.9 . Most paMean valid accelerometer wear time differed significantly pre- and post-surgery, and participants wore the accelerometer 30 min longer per day post-surgery. We do not know the reason for these differences, but a large weight loss may result in being out of bed for longer during the day. Another reason could be a better routine to wear the accelerometer at the follow-up. Longer wear time will likely result in longer sedentary time, as people in general spend more than half of their waking time sedentary . When coPersons with obesity often report that they are not used to performing PA and do not identify as someone who practices PA , 38, whiFindings in the current and previous studies indicate that MBS is not sufficient to change a person’s PA habits. There is a need for further research to understand what motivates patients post-MBS to decrease sedentary time and perform PA of higher intensities and how best to support them. Previous studies show that this objective can be difficult to achieve. Neither group-based educational programs of four to ten sessions, nor a motivational individual PA intervention for 6 months resulted in increased PA –41. An iThe major strengths of this study include the longitudinal design with objective monitoring of PA with accelerometers, as well as the investigation of mediators for PA. It is one of few studies using validated measures of enjoyment, self-efficacy for exercise, and social support in relation to PA post-MBS.The power calculation indicated a sample size of 100 participants to achieve a power of 94%. However, the SD of sedentary time was lower in our study (63.2 min/day) than in the study the power calculation was derived from (141.5 min/day), and the power in our study was as strong as in the power calculation.A major limitation of our study is the large dropout rate. At follow-up, 67% of the participants completed the questionnaires, but only 56% had also valid accelerometer data. It was a challenge to measure PA in individuals in free-living conditions. Despite intensive monitoring, several accelerometers were not returned, resulting in complete follow-up measures from 50 participants. Therefore, the results need to be interpreted with some caution. A higher proportion of dropouts were operated with SG; otherwise, we found no significant differences between completers and dropouts.Our study population of 90 participants represents almost 20% of all persons who underwent MBS at the university hospital during the study period. Due to organizational reasons and periods of lack of personnel at the surgery unit, only 147 of 455 operated patients were asked for consent to participate in the study. It is possible that persons who were not asked or declined participation were less physically active, which may have biased our results.We assessed outcomes 12 to 18 months after MBS, and participants may have experienced the “honeymoon effect” with major weight loss and maximum motivation at this point of time. Effects also need to be evaluated in the longer term to determine whether the changes are sustainable over time.Results in the present study show a significant increase in enjoyment of, and self-efficacy and social support for, PA. Light PA and total PA increased, and sedentary time as percentage of wear time decreased, but the increase in mediators for PA did not result in an increase in MVPA on group level.ESM 1(DOCX 31 kb)


The application of vaterite microparticles for mucosal delivery depends on their interaction with mucin and immune cells. As we have shown previously, the binding of mucin onto particles enhances the generation of reactive oxygen species by neutrophils. The attenuation of the pro-oxidant effect of the bound mucin through the modification of vaterite could improve its biocompatibility. Hybrid microparticles composed of vaterite and pectin (CCP) were prepared using co-precipitation. In comparison with vaterite (CC), they had a smaller diameter and pores, a greater surface area, and a negative zeta-potential. We aimed to study the cytotoxicity and mucin-dependent neutrophil-activating effect of CCP microparticles. The incorporated pectin did not influence the neutrophil damage according to a lactate dehydrogenase test. The difference in the CC- and CCP-elicited luminol or lucigenin chemiluminescence of neutrophils was insignificant, with no direct pro- or antioxidant effects from the incorporated pectin. Unlike soluble pectin, the CCP particles were ineffective at scavenging radicals in an ABAP–luminol test. The fluorescence of SYTOX Green demonstrated a CCP-stimulated formation of neutrophil extracellular traps (NETs). The pre-treatment of CC and CCP with mucin resulted in a 2.5-times-higher CL response of neutrophils to the CC-mucin than to the CCP-mucin. Thus, the incorporation of pectin into vaterite microspheres enabled an antioxidant effect to be reached when the neutrophils were activated by mucin-treated microparticles, presumably via exposed ligands. Vaterite is a polymorph of calcium carbonate, with particle dimensions ranging from nanometres to tens of micrometres, a spherical surface, a highly developed internal structure, and a high porosity; it is biocompatible and biodegradable but thermodynamically unstable .Vaterite has prominent mucoadhesive properties  suitable•+ and DPPH• or hydroxyl radicals, depending on pectin’s structure, and the activity was about 200-times lower in comparison with ferulic acid or Trolox [In the present study, vaterite was co-precipitated with apple pectin, resulting in hybrid microparticles. Commercially available pectin from apple pulp is a partially methyl-esterified and non-sulphated polysaccharide. Its main chain is composed of 1,4-linked α-D-galactosyluronic acid and α-1,2-L-rhamnose, and its side chains include various uncharged monosaccharides such as arabinose, galactose, and others . Pectin r Trolox .Pectin has been studied as a component of delivery systems and is incorporated into gels, films, and polyelectrolyte complexes with alginate, chitosan, casein, gelatine, carboxymethylcellulose , inorganIn order to study some aspects of the biocompatibility of hybrid vaterite–pectin microparticles, we evaluated their antioxidant properties, cytotoxicity, sorption of mucin, and activation of the neutrophil respiratory burst. The antioxidant effects of pectin and CCP were evaluated in a model system of ABAP–luminol, as well as at the activation of neutrophils in vitro, in comparison with CC. Mucin was adsorbed onto the particles or added into the measurement media. Since the neutrophil oxidative burst assayed with chemiluminescence could be affected by cell viability, we also assessed the cytotoxicity of the particles using the extracellular lactate dehydrogenase (LDH) activity, the formation of NETs using SYTOX Green fluorescence, and the functional activity as the amplitude of the chemiluminescent response to the second activator, phorbol 12-myristate 13-acetate (PMA).• in the presence of oxygen. Peroxyl radicals were detected using luminol-enhanced chemiluminescence (Lum-CL) [The antioxidant activity of pectin was evaluated using the thermally decomposing compound 2,2′-azobis(2-amidinopropane) dihydrochloride (ABAP), giving peroxyl radicals ROO(Lum-CL) . AntioxiWith the growth of the pectin concentration, the lag-period increased a,b as a − ions using an iodometric titration - or lucigenin (Luc)-CL of neutrophils a,b.The inhibition of spontaneous Lum-CL by pectin was stronger than the inhibition of Luc-CL, while the PMA-stimulated responses decreased proportionally. These effects of 1 mg/mL of pectin were not caused by neutrophil damage, according to the LDH test, and could be attributed to the scavenging of superoxide radicals (registered using Luc-CL) and hypochlorite (registered using Lum-CL) by pectin.w/w. In comparison with CC, the percentage of calcite in the CCP microparticles was the same, but the diameter of the CCP microspheres was 1.8-times smaller , in contrast to CC. This indicated that the incorporated pectin was partially exposed on the surface of the CCP microparticles.In order to evaluate the antioxidant activity of the incorporated pectin, we also used an ABAP–luminol system (like in the experiments with pectin), but no effects of CCP (or CC) microparticles in concentrations up to 1 mg/mL were registered. Indeed, this concentration corresponded to no more than 50 μg/mL of pectin, which was below the sensitivity of the method according to The incorporated pectin reduced the adsorption of mucin by the CCP microparticles by 1.7-fold in comparison with CC . Mucin fMicroparticles of 2–4 μm in diameter are microbe-sized objects whose size facilitates their interaction with neutrophils, depending on the zeta-potential and the exposed ligands for neutrophil receptors. The generation of ROS/RHS was detected using Luc-CL sensitive to superoxide anions and Lum-CL in order to detect mainly hypochlorite. With regards to the potential effects of mucin adsorption, the CL was also measured in the presence of 0.5 mg/mL of mucin, and/or neutrophils were stimulated with CC and CCP microparticles pre-treated with mucin (CC-M and CCP-M).There were no significant differences in the intensity of the neutrophil Lum-CL responses to the CC and CCP microparticles, even when mucin was added to the medium b, and thp ˂ 0.05).The pre-treatment of CC microparticles with mucin enhanced both the Lum-CL b and LucA comparison of the amplitude values of the PMA-stimulated CL after the activation of neutrophils with microparticles showed that only CCP-M caused a decrease in the Luc-CL value by 15% e, with n− .The pre-treatment of the CC and CCP microparticles with OClIt is known that ROS and/or RHS can stimulate activated cells to form NETs and release DNA decorated with histones, elastase, myeloperoxidase (MPO), and other proteins . Since pNET formation was measured using the SYTOX Green staining of extracellular DNA a and thePectin (1 mg/mL in solution) significantly decreased the percentage of SG-positive neutrophils under the stimulation of neutrophils with PMA, a potent inducer of NETosis a, as welMoreover, unlike PMA, CC- and CCP-induced NET formation was not inhibited by soluble pectin (1 mg/mL), even after mucin adsorption: in the presence of pectin, F504/523 increased to 133 ± 23% for CCP, but did not change for CCP-M (102 ± 16%). For CC and CC-M, the effects were more significant: 250 ± 79% and 219 ± 35%, respectively.The development of new vehicles for mucosal delivery requires the synthesis of multifunctional particles composed of components that not only play structural roles, but also have other valuable properties. Our study was aimed at evaluating the biocompatibility of pectin–vaterite hybrid microparticles as a mucosal drug-delivery system. They can be used for intranasal  and pero•, ABTS•+, and •OH, registered in the range of 0.7–10 mg/mL [Among the potential polysaccharides for incorporation into inorganic particles, pectin has attracted growing attention from researchers. The antioxidant properties of pectin are usually evaluated as radical-scavenging activity towards DPPH10 mg/mL ,20.50 of 0.6 mg/mL  ,27. Pectin layer . At the h CC-OCl .No cytotoxic or cell-inactivating effects of the incorporated pectin were found under our experimental conditions. However, there was a difference between the CC and CCP microparticles in the induction of NETosis. Free pectin significantly decreased the formation of NETs stimulated with PMA, presumably via its antioxidant activity. However, CCP microparticles were more capable as inducers of NETosis than CC, and since the neutrophils remained alive and functionally active, this could be characterised as a vital NETosis . MoreoveIt is difficult to predict the in vivo benefits of vaterite microparticles modified with pectin (CCP) on the basis of in vitro experiments, but there are some points that look encouraging. Thus, with regard to the development of new drug-delivery systems, the hybrid pectin–vaterite microparticles demonstrated a number of valuable properties. Vaterite and pectin are natural materials, with both being biocompatible and biodegradable ,31,32,33We have shown that, in line with vaterite microparticles , hybrid In the absence of mucin in the measurement buffer, there was no difference between CC and CCP in their activating ability towards neutrophils b. With m2 (≥93.0%), Na2CO3 (≥99.0%), CaCl2, NaCl, EDTA, Tris, Histopaque , DMSO, NaOCl, FITC, phorbol 12-myristate 13-acetate (PMA), 2,2′-azobis(2-amidinopropane)dihydrochloride (ABAP), luminol, lucigenin, mucin from a porcine stomach , pectin from apples (methyl-esterified), sodium L-ascorbate, and all salts and solvents for the preparation of solutions were purchased from Sigma-Aldrich . The Krebs–Ringer buffer solution was purchased from Merck . The Olvex-LDH kit was purchased from LLC Olveks Diagnosticum, SPb, Russia. The SYTOX Green stain was purchased from ThermoFisher Scientific .CaClThe anti-MPO antibodies conjugated with Cy5 were kindly provided by A.V. Sokolov and N.P. Gorbunov. The fluorescein isothiocyanate-labelled mucin (FITC-mucin) (17 ± 2% modified amino groups) was kindly provided by N.G. Balabushevich.2 and Na2CO3 in 0.05 M Tris buffer (pH 7.0) as described earlier [Vaterite microparticles (CC) were synthesized by mixing solutions of CaCl earlier . For theparticles) was calculated using the Gwiddion software version 2.0.For scanning electron microscopy, the microparticles were deposited onto silicon wafers and characterised using a Zeiss Merlin microscope . The SEM accelerating volage was 1–2 kV. The diameter of the microspheres . The specific surface area was estimated using the BET method, and the pore size was calculated using the Barrett–Joyner–Halenda (BJH) method.The ζ-potential of the biopolymers and microparticles and the hydrodynamic diameter of mucin was measured using a Zetasizer Nano ZS analyser  with He-Ne laser, 5 mW, 633 nm, and estimated using the Smoluchowski equation. The Universal “Dip” cell (ZEN1002) was applied to measure the ζ-potential.An X-ray powder diffraction analysis was performed using a Miniflex 600  with a silicon drift detector on powdered samples using Cu Ka 1.5418 Å radiation and a Ni filter with 2ɵ from 20° to 60°.For the assay of the adsorption of mucin, a mixture of 40 mg/mL of microparticles and 1 mg/mL of mucin from a porcine stomach in 0.05 M Tris buffer (pH 7.4) was incubated for 30 min at 25 °C. Then, the particles were precipitated using centrifugation (5 min at 5000 rpm). The residual concentration of mucin in the supernatants was assayed using optical absorbance at 214 nm or 260 nm. In the experiments with CCP microparticles, mucin was assayed with gel filtration using a BioFox 17 SEC column  as described earlier [Fluorescent images of the microparticles treated with FITC-labelled mucin (M-FITC) were obtained using a Nicon Ni-E microscope , with a filter FITC .The antioxidant activity of free pectin and the microparticles was assayed using the Lum-CL, measured using the luminometer Lum 1200 .A total of 1 mL of measurement media, namely 50 μM luminol in 50 mM Tris-HCl buffer with 0.15 M NaCl and 1 mM EDTA (pH 8.5), was incubated in a luminometer cell for 5 min at 37 °C. Then, ABAP was added up to 1 mM. When chemiluminescence developed, pectin up to 2 mg/mL or microparticles up to 1 mg/mL were added, the solution was mixed quickly, and the CL was registered in continuous mode. The antioxidant effect was evaluated by the duration of the lag-period (Lag) after sample injection. Sodium ascorbate was used as a standard antioxidant.Human neutrophils were isolated from the normal blood of 6 volunteers aged 35–60 years without inflammatory diseases, on the basis of their informed consent and agreement and approved by the local ethics committee. The blood was layered over a double gradient of Histopaque (1.077/1.119 g/L), and after centrifugation for 45 min, the neutrophils were collected, washed, and resuspended in Krebs–Ringer solution. The cell concentration was assayed by direct counting using a Goryaev chamber, with a neutrophil purity of >95%.6 cells/mL). Microparticles and pectin were added in a concentration of 1 mg/mL if not otherwise indicated. If necessary, the measurement medium was supplemented with 0.5 mg/mL of mucin. The intensity of the CL response was assayed as an integral value for 20–40 min and presented as a percentage of the control values (without particles or pectin). When the CL maximum was reached, some cuvettes were centrifugated at 2000 rpm for 5 min, and the supernatants were collected for a further assay of the LDH activity and SYTOX Green fluorescence. PMA (0.3 μM) was added to the other cuvettes at the same moment and the amplitude of the CL was measured.The CL was measured in 0.5 mL of Krebs–Ringer solution (pH 7.4) with 0.2 mM luminol (Lum-CL) or lucigenin (Luc-CL) and neutrophils .Microparticles (10 mg/mL) were mixed in an equal volume with 100 μM NaOCl (pH 7.0) and incubated for 30 min at 37 °C with mild shaking. Then, the particles were precipitated at 5000 rpm for 5 min, washed twice, and resuspended in 0.15 M NaCl.The cytotoxicity assay was based on an evaluation of the activity of extracellular LDH, by measuring the rate of NADH utilisation using an Olvex-LDH kit.A total of 5 μL of 50 μM SYTOX Green in DMSO was added to 500 μL of supernatant obtained after the CL analysis (p. 4.6) and incubated for 15 min at 25 °C. Then, an equal volume of Krebs–Ringer solution was added and the F504/F523 fluorescence was measured using a solar fluorometer .The formation of neutrophil extracellular traps (NETosis) was detected using flow cytometry as described elsewhere . Monoclot-test using STATISTICA software version 12 . p-values ˂ 0.05 were assumed to be significant. The data are represented as the mean value ± standard deviation.The statistical significance levels were calculated with Student’s